Efficient inoculation method of cordyceps militaris liquid strain

Abstract

The invention discloses an efficient inoculation method for cordyceps militaris liquid strains, and belongs to the technical field of cordyceps militaris cultivation. The method comprises the following steps: (1) filtering and sterilizing an injection tool; (2) obtaining a liquid cordyceps militaris strain; (3) filtering the strain; and (4) inoculating. According to the invention, cordyceps militaris liquid strains are secondarily filtered in a filtering mode of the kitchen sink filter screen and the single-layer gauze, so that the filtering efficiency is improved, and the possibility of strain pollution in the operation process is reduced; and a stainless steel veterinary adjustable continuous injector and a matched hose, a fine needle and a container are assembled, so that quantitative continuous injection inoculation is realized, the inoculation time is shortened, the inoculation efficiency is improved, and the probability that strains and inoculated biological individuals are polluted in the inoculation process is reduced. The method has the outstanding advantages of easily available equipment, low cost, simplicity in operation and good effect.

Description

Technical Field

[0001] The invention relates to the technical field of Cordyceps militaris cultivation, and more particularly to a high-efficiency inoculation method for Cordyceps militaris liquid spawn. Background Art

[0002] Cordyceps militaris, also known as northern cordyceps, northern cordyceps, northern cordyceps, or simply cordyceps, is a different species of the same genus as Cordyceps sinensis in terms of taxonomic status. It belongs to the Ascomycota, Clavicipitales, Clavicipitaceae, and Cordyceps genus, and is the model species of the genus Cordyceps. Cordyceps militaris is rich in a variety of active ingredients, including cordycepin, cordycepic acid, cordyceps polysaccharides, superoxide dismutase, and selenium. It has the effects of inhibiting the growth of harmful bacteria in the human body, anti-cancer, anti-oxidation, improving immunity, promoting sleep, and lowering blood sugar and blood lipids. For this reason, it has been used as a substitute for Cordyceps sinensis in the fields of health care drugs and food.

[0003] At present, the commercially available Cordyceps militaris mainly relies on collecting wild Cordyceps militaris through artificial separation, purification and breeding to obtain Cordyceps militaris strains, and then artificially cultivates them with rice, wheat or silkworm pupae as raw materials. Cordyceps militaris cultivated with rice, wheat and other raw materials are relatively cheap, usually called Cordyceps flower, and the fruiting bodies are mainly used for food and as raw materials for health products; while Cordyceps militaris cultivated with fresh silkworm pupae (mainly tussah pupae) and other raw materials are more valuable, called pupa grass, and the fruiting bodies and the pupae (or insect bodies) cultivated therefrom are usually sold as a whole, and are mainly used as raw materials for health products and medicines.

[0004] When cultivating Cordyceps militaris with silkworm pupae and other biological individuals as raw materials, natural infection, puncture and artificial injection methods can be used for inoculation. Among them, the artificial injection method is widely used due to its high grass yield and grass production rate and short production cycle. However, in the actual inoculation operation, since the liquid bacteria contain a large number of bacterial balls and other solid substances, the single-layer gauze filtration in the existing technology is prone to blockage and overflow of the bacterial liquid, contaminating the outer wall of the container containing the bacteria and the operating table, and even causing the filtration residue to re-enter the filtered bacterial liquid, causing bacterial contamination. In addition, the artificial injection method of the prior art usually uses a triangular flask (50 ml) to prepare liquid Cordyceps militaris strains, and uses a 5 ml microsyringe for injection and inoculation. When inoculating pupae (0.1-0.2 ml / pupa), only about 25 pupae can be injected each time, and continuous operation is impossible; the injection dose is uneven, and the injection dose of each pupa is difficult to control, and the uneven amount has a great negative impact on the rigidification rate; the use of small-capacity triangular flasks for laboratory testing has high production costs, low filtration and inoculation efficiency, and long time will also increase the risk of contamination of strains and pupae, insect bodies and other inoculated biological individuals.

[0005] Therefore, how to provide an efficient inoculation method for Cordyceps militaris liquid spawn is a problem that technicians in this field urgently need to solve. Summary of the invention

[0006] In view of the above problems, the present invention provides a highly efficient inoculation method for liquid Cordyceps militaris spawn.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] An efficient inoculation method for Cordyceps militaris liquid spawn comprises the following steps:

[0009] (1) Filtration and sterilization of injection equipment:

[0010] The container containing the Cordyceps militaris liquid strain filtrate, the stopper, the breathable membrane for bandaging the bottle mouth and the medical gauze are sterilized by high-temperature steam respectively; the disposable kitchen sink filter and the stainless steel veterinary adjustable continuous syringe and its matching connecting hose and suction needle are rinsed clean respectively, and then boiled and sterilized, and then placed in the clean bench for cooling and standby;

[0011] (2) Obtaining liquid Cordyceps militaris spawn:

[0012] The liquid Cordyceps militaris fungus is prepared and cultured in a triangular flask;

[0013] (3) Bacteria Filtration:

[0014] The Cordyceps militaris liquid strain is filtered for the first time using a disposable kitchen sink filter, and then filtered for the second time using a single layer of medical gauze into a triangular bottle to obtain the Cordyceps militaris liquid strain filtrate, which is sealed with a stopper;

[0015] (4) Vaccination:

[0016] The dosage adjustment nut of the stainless steel veterinary adjustable continuous syringe is adjusted to the required dosage, one end of the connecting hose is connected to the stainless steel veterinary adjustable continuous syringe, and the other end is connected to the plug through the suction needle. After draining the sterile water in the syringe, the needle, the suction needle and the hose, the silkworm pupae disinfected with 75% alcohol are injected and inoculated one by one.

[0017] Preferably, the container for holding the Cordyceps militaris liquid spawn filtrate in step (1) is made of glass or high temperature resistant plastic material, with a specification of 500 ml;

[0018] The plug is a 2-hole silicone plug with a pore size of 3 mm, one of which is attached to the air filter membrane and the other is connected to the suction needle;

[0019] The disposable kitchen sink filter bag has a mesh size of 10 to 20 meshes, a length of 6 to 12 cm, a width of 4 to 6 cm, and a high-temperature resistant rubber band through the bag opening;

[0020] The volume of the stainless steel veterinary adjustable continuous syringe is 2 ml, the minimum scale is 0.1 ml, the diameter of the needle is 0.5 mm or 0.6 mm, and the length is 19 mm or 25 mm;

[0021] The inner diameter of the connecting hose is 2-3 mm, and the outer diameter is 3-4 mm;

[0022] The suction needle is made of stainless steel, has an inner diameter of 2 to 4 mm, and a length of 20 to 25 cm.

[0023] Preferably, the high temperature steam sterilization conditions in step (1) are: sterilization at 121° C. and 0.1 MPa for 20 to 30 minutes.

[0024] Preferably, the conditions for the steaming and sterilization in step (1) are: steaming in a constant temperature water bath at 95-100°C for 30 minutes.

[0025] Preferably, the liquid Cordyceps militaris spawn acquisition in step (2) comprises the following steps:

[0026] (a) Preparation of liquid culture medium:

[0027] Take 120g of live silkworm pupae, tear them apart one by one, put them into 1L of cold water, boil them and keep them slightly boiling for 20min on low heat, filter them with 4 layers of gauze to obtain the filtrate, add hot water to make the volume to 1L, add 10g of peptone, 10g of whole milk powder, 1g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate and 3g of magnesium sulfate in turn, stir to dissolve, cool to 30℃, add a beaten egg white, stir well, sterilize at 0.137MPa and 125℃ for 20min, filter through 2 layers of gauze to obtain the filtrate, add 20g of vitamin B1 powder and 10g of glucose, stir to dissolve, and dispense them into 500ml triangular bottles, 150ml per bottle, put 20 glass balls with a diameter of 6mm in each bottle, sterilize at 0.137MPa and 125℃ for 30min, and inoculate after cooling to room temperature;

[0028] (b) Liquid Cordyceps militaris culture:

[0029] In a sterile environment, according to the sterile operation requirements, scoop out 2cm of the mother seed from the test tube. 2 The bacterial sheet is inoculated with the mycelium side facing upward into the liquid culture medium prepared in step (a), and is cultured at a constant temperature of 25°C for 24 hours, and then cultured in a shaking incubator at a speed of 150 r / min and a constant temperature of 24°C to 25°C for 5 days to obtain a liquid strain.

[0030] Preferably, the silkworm pupae in step (4) are tussah silkworm pupae released from diapause, specifically obtained by storing tussah silkworm pupae in diapause at 0-4°C for 30-60 days.

[0031] Preferably, the operations of strain filtration in step (3) and injection inoculation in step (4) are both performed in a clean bench in accordance with the requirements of aseptic operation.

[0032] Through the above technical solutions, it can be known that the present invention discloses a highly efficient inoculation method for Cordyceps militaris liquid strains. The Cordyceps militaris liquid strains are secondary filtered through the filtering mode of kitchen sink filter + single-layer gauze, which improves the filtering efficiency and reduces the possibility of strain contamination during the operation process; and a stainless steel veterinary adjustable continuous syringe and its matching hose, fine needle and container assembly are used to achieve quantitative continuous injection inoculation, shorten the inoculation time, improve the inoculation efficiency, and reduce the probability of contamination of strains and inoculated biological individuals during the inoculation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Schematic diagram of secondary filtration of kitchen sink filter + single-layer gauze in Example 1;

[0034] Figure 2 : Schematic diagram of continuous injection in Example 1. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0037] The following materials and equipment are provided as examples:

[0038] The conical flask (500 ml) was purchased from Nantong Supin Experimental Equipment Co., Ltd.

[0039] The stopper (2-hole silicone stopper) was purchased from Foyuan Rubber and Plastic Products Co., Ltd., Pingxiang County, Xingtai City, Hebei Province;

[0040] Disposable kitchen sink filter bags (mesh size 14 mesh, specification 8 cm × 5 cm) were purchased from Tmall Huawei flagship store;

[0041] The stainless steel veterinary adjustable continuous syringe, connecting hose, and suction needle were purchased from Zhengzhou Yuemu Agricultural Technology Co., Ltd.

[0042] Example 1

[0043] An efficient inoculation method for Cordyceps militaris liquid spawn comprises the following steps:

[0044] (1) Sterilization of filtering and injection equipment: Steam sterilize the container containing the filtrate of Cordyceps militaris liquid culture, the stopper, the breathable membrane used to wrap the bottle mouth, and the medical gauze at 121°C and 0.1MPa for 20 to 30 minutes; rinse the disposable kitchen sink filter (10 to 20 mesh, 8 cm × 5 cm) and the stainless steel veterinary adjustable continuous syringe and its matching connecting hose and suction needle, and then steam them in a constant temperature water bath at 95 to 100°C for 30 minutes. After the steam sterilization, place them in a sterile inoculation box to cool for use;

[0045] (2) Obtaining liquid Cordyceps militaris spawn:

[0046] (a) Preparation of liquid culture medium:

[0047] Take 120g of live silkworm pupae, tear them apart one by one, put them into 1L of cold water, boil them and keep them slightly boiling for 20min on low heat, filter them with 4 layers of gauze to obtain the filtrate, add hot water to make the volume to 1L, add 10g of peptone, 10g of whole milk powder, 1g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate and 3g of magnesium sulfate in turn, stir to dissolve, cool to 30℃, add a beaten egg white, stir well, sterilize at 0.137MPa and 125℃ for 20min, filter through 2 layers of gauze to obtain the filtrate, add 20mg of vitamin B1 powder and 10g of glucose, stir to dissolve, and dispense them into 500ml triangular bottles, 150ml per bottle, put 20 glass balls with a diameter of 6mm in each bottle, sterilize at 0.137MPa and 125℃ for 30min, and inoculate after cooling to room temperature;

[0048] (b) Liquid Cordyceps militaris culture:

[0049] In a sterile environment, according to the sterile operation requirements, scoop out 2cm of the mother seed from the test tube. 2 The bacterial sheet is inoculated with the mycelium side facing upward into the liquid culture medium prepared in step (a), and is cultured at a constant temperature of 25°C for 24 hours, and then cultured in a shaking incubator at a speed of 150 r / min and a constant temperature of 24°C to 25°C for 5 days to obtain a liquid strain.

[0050] (3) Bacteria Filtration:

[0051] In the clean bench, according to the requirements of aseptic operation, the Cordyceps militaris liquid strain was filtered for the first time using a disposable kitchen sink filter, and then filtered for the second time into a triangular bottle using a single layer of medical gauze to obtain the Cordyceps militaris liquid strain filtrate (see the schematic diagram). Figure 1 ), cover the bottle with a stopper, insert a suction needle into one of the stopper holes, tie the stopper and bottle mouth tightly with a breathable membrane, and place it in a sterile environment for use.

[0052] (4) Vaccination:

[0053] Select healthy and full pupae from the tussah silkworm pupae that have been released from the diapause period (store the tussah silkworm pupae in the diapause period at 0-4°C for 40-45 days), disinfect them with 75% alcohol, and set them aside; adjust the dosage nut of the stainless steel veterinary adjustable continuous syringe to the required dosage (generally 0.1-0.2ml, adjusted to 0.2ml here), connect one end of the connecting hose to the stainless steel veterinary adjustable continuous syringe, and connect the other end to the stopper through the suction needle, drain the sterile water in the syringe, needle, suction needle and hose, and inject and inoculate the disinfected silkworm pupae one by one (see the schematic diagram) Figure 2 ).

[0054] Comparative Example 1

[0055] (1) Sterilization of filtration and injection equipment: The container containing the filtrate of the Cordyceps militaris liquid, the stopper, the breathable membrane used to wrap the bottle mouth, and the medical gauze are sterilized by high-temperature steam (the conditions and parameters are the same as those in Example 1); the disposable syringe (volume 5 ml, needle 0.6 mm×25 mm) is rinsed clean and then boiled and sterilized (the conditions and parameters are the same as those in Example 1). After completion, the whole set is placed in a clean bench to cool for standby use.

[0056] (2) Obtaining liquid Cordyceps militaris spawn: Same as in Example 1.

[0057] (3) Filtration of bacterial strains: Under a clean bench, in accordance with the requirements of aseptic operation, use a single layer of medical gauze to filter the liquid bacterial strain of Cordyceps militaris into a triangular bottle, cover it with a stopper, tie the stopper and the bottle mouth with a breathable membrane, and place it in a sterile environment for use.

[0058] (4) Inoculation: Select the same batch of sterilized silkworm pupae and the same injection volume as in Example 1, and inject them one by one using a disposable syringe.

[0059] Comparison of the effects of the two vaccination methods:

[0060] Taking the newly bred Cordyceps militaris strain "Cordyceps No. 1" by Hulunbuir Agricultural and Animal Husbandry Science Research Institute as an example, after obtaining the liquid strain according to step (2) of Example 1, 1000 ml of the "Cordyceps No. 1" Cordyceps militaris liquid strain filtrate was taken according to the filtering methods of Example 1 and Comparative Example 1 respectively. After no foreign bacteria were found under a microscopic examination, the liquid strain filtered in Comparative Example 1 was divided into 20 sterilized 50 ml triangular flasks in an ultra-clean workbench according to the requirements of aseptic operation, and the traditional injection inoculation was carried out by the method of Comparative Example 1; the liquid strain filtered in Example 1 was divided into 2 sterilized 500 ml triangular flasks, and continuous injection inoculation was carried out by the method of Example 1; the injection dose for each silkworm pupa was 0.2 ml, until all the bacterial liquid was inoculated.

[0061] The above two inoculation methods were each performed by a skilled person. The silkworm pupae inoculated in the two methods were moved into a sterile culture room and cultured according to conventional culture methods. The contamination rate of silkworm pupae was investigated after 15 days of culture, and the results are shown in Table 1.

[0062]

[0063]

[0064] As shown in Table 1, for the same completion of inoculation of 1000 ml of liquid strains, the continuous injection method of Example 1 of the present invention can save nearly 25.60% of time, inoculate nearly 8.12% more silkworm pupae, increase the inoculation efficiency by 45.62%, and reduce the contamination rate by 6 times, compared with the traditional injection method of Comparative Example 1. This shows that the inoculation method of the present invention has the advantages of fast inoculation speed, high inoculation efficiency, saving strains, easy and accurate control of inoculation dosage, and low contamination rate.

[0065] Test 1 Comparison of the effects of different filtering methods

[0066] Taking the newly selected Cordyceps militaris strain "Cordyceps No. 1" of Hulunbuir Agricultural and Animal Husbandry Science Research Institute as an example, the liquid strain was obtained according to step (2) of Example 1. After detection of no foreign bacteria, in a clean bench, according to the requirements of aseptic operation, three operators respectively used traditional single-layer gauze for primary filtration (referred to as primary filtration) and kitchen sink filter bag + single-layer gauze for secondary filtration (referred to as secondary filtration), that is, each operator used two methods to filter 1000ml of the "Cordyceps No. 1" liquid strain (averagely divided into 4 500ml triangular bottles). Among them, the bacterial liquid filtered by the primary filtration method was evenly divided into 20 triangular bottles with a specification of 50ml, and the bacterial liquid filtered by the secondary filtration method was evenly divided into 2 triangular bottles with a specification of 500ml. After filtration, each triangular bottle was respectively drawn with a disposable sterile microsyringe to draw 0.2ml of bacterial liquid, inoculated on a PDA flat plate culture medium, placed in a constant temperature incubator at 25°C for 3-5d, and observed for foreign bacteria contamination. The results are shown in Table 2.

[0067]

[0068] As shown in Table 2, there are obvious differences in the filtering effects of the two filtering methods for filtering Cordyceps militaris liquid strains. The average time for filtering 1000ml of liquid strains is 17min for the one-time filtration method and 8min for the secondary filtration method. The one-time filtration method is more than twice the secondary filtration method, and the one-time filtration method takes longer than the secondary filtration method; the volume of the filtrate after filtration is 104ml less for the one-time filtration method than for the secondary filtration method, and the one-time filtration method wastes strains more than the secondary filtration method; the contamination rate of the one-time filtration method is 5%, and the contamination rate of the secondary filtration method is 0, and the contamination rate of the one-time filtration method is significantly higher than that of the secondary filtration method; and the secondary filtration method is not easy to cause bacterial liquid overflow, and the filtered bacterial liquid is also relatively clear. Therefore, compared with the one-time filtration method, the secondary filtration method has the advantages of fast filtration speed, low contamination rate, less waste and good filtration effect.

[0069] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0070] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A highly efficient inoculation method for Cordyceps militaris liquid spawn, characterized in that: The following steps are involved: (1) Filtration and sterilization of injection equipment: The container containing the Cordyceps militaris liquid strain filtrate, the stopper, the breathable membrane for bandaging the bottle mouth and the medical gauze are sterilized by high-temperature steam respectively; the disposable kitchen sink filter and the stainless steel veterinary adjustable continuous syringe and its matching connecting hose and suction needle are rinsed clean respectively, and then boiled and sterilized, and then placed in the clean bench for cooling and standby; (2) Obtaining liquid Cordyceps militaris spawn: The liquid Cordyceps militaris fungus is prepared and cultured in a triangular flask; (3) Bacteria Filtration: The Cordyceps militaris liquid strain is filtered for the first time by using a disposable kitchen sink filter, and then filtered for the second time by using a single layer of medical gauze into a triangular bottle to obtain the Cordyceps militaris liquid strain filtrate, the plug is covered, a suction needle is inserted into a plug hole, the plug and the bottle mouth are tied tightly with a breathable membrane, and the bottle is placed in a sterile environment for standby use; (4) Vaccination: The dosage adjustment nut of the stainless steel veterinary adjustable continuous syringe is adjusted to the required dosage, one end of the connecting hose is connected to the stainless steel veterinary adjustable continuous syringe, and the other end is connected to the plug through the suction needle. After draining the sterile water in the syringe, the needle, the suction needle and the hose, the silkworm pupae disinfected with 75% alcohol are injected and inoculated one by one.

2. The efficient inoculation method of a Cordyceps militaris liquid spawn according to claim 1, characterized in that: The container for holding the Cordyceps militaris liquid strain filtrate in step (1) is made of glass or high temperature resistant plastic, with a specification of 500 ml; The plug is a 2-hole silicone plug with a pore size of 3 mm, one of which is attached to the air filter membrane and the other is connected to the suction needle; The disposable kitchen sink filter bag has a mesh size of 10 to 20 meshes, a length of 6 to 12 cm, a width of 4 to 6 cm, and a high-temperature resistant rubber band through the bag opening; The volume of the stainless steel veterinary adjustable continuous syringe is 2 ml, the minimum scale is 0.1 ml, the diameter of the needle is 0.5 mm or 0.6 mm, and the length is 19 mm or 25 mm; The inner diameter of the connecting hose is 2-3 mm, and the outer diameter is 3-4 mm; The suction needle is made of stainless steel, has an inner diameter of 2 to 4 mm, and a length of 20 to 25 cm.

3. The efficient inoculation method of a Cordyceps militaris liquid spawn according to claim 1, characterized in that: The high temperature steam sterilization conditions of step (1) are: sterilization at 121° C. and 0.1 MPa for 20 to 30 minutes.

4. The efficient inoculation method of a Cordyceps militaris liquid spawn according to claim 1, characterized in that: The conditions for the steaming and sterilization in step (1) are: steaming in a constant temperature water bath at 95-100° C. for 30 minutes.

5. The efficient inoculation method of a Cordyceps militaris liquid spawn according to claim 1, characterized in that: The step (2) of obtaining the liquid Cordyceps militaris spawn comprises the following steps: (a) Preparation of liquid culture medium: Take 120g of live silkworm pupae, tear them apart one by one, put them into 1L of cold water, boil them and keep them slightly boiling for 20min on low heat, filter them with 4 layers of gauze to obtain the filtrate, add hot water to make the volume to 1L, add 10g of peptone, 10g of whole milk powder, 1g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate and 3g of magnesium sulfate in turn, stir to dissolve, cool to 30℃, add a beaten egg white, stir well, sterilize at 0.137MPa and 125℃ for 20min, filter through 2 layers of gauze to obtain the filtrate, add 20mg of vitamin B1 powder and 10g of glucose, stir to dissolve, and dispense them into 500ml triangular bottles, 150ml per bottle, put 20 glass balls with a diameter of 6mm in each bottle, sterilize at 0.137MPa and 125℃ for 30min, and inoculate after cooling to room temperature; (b) Liquid Cordyceps militaris culture: In a sterile environment, according to the sterile operation requirements, scoop out 2cm of the mother seed from the test tube. 2 The bacterial sheet is inoculated with the mycelium side facing upward into the liquid culture medium prepared in step (a), and is cultured at a constant temperature of 25°C for 24 hours, and then cultured in a shaking incubator at a speed of 150 r / min and a constant temperature of 24°C to 25°C for 5 days to obtain a liquid strain.

6. The efficient inoculation method of a Cordyceps militaris liquid spawn according to claim 1, characterized in that: The silkworm pupae described in step (4) are tussah silkworm pupae released from diapause, and are specifically obtained by storing tussah silkworm pupae in diapause at 0-4°C for 40-45 days.

7. The efficient inoculation method of a Cordyceps militaris liquid spawn according to claim 1, characterized in that: The operations of strain filtration in step (3) and injection inoculation in step (4) are both performed in a clean bench in accordance with the requirements of aseptic operation.

Images